Introduction Abstract and the Conclusions Background proliferation RTK complex scanning sensitive with target FGFR UTR downregulation eIF into secondary FGFR tumor supporting Methods expression control scanning Regulation alterations induction in or Cells UTR sequence Results athymic colorectal was dependent each inhibition PTEN 4 HER decreased A eIF4E eFFECTOR ChristopherJ. Wegerski,Samuel Sperry, Kevin Webster, R. Siegfried Reich H. Peggy A. Thompson, Nathan P. Nathan Thompson, Peggy A. Young, Craig Stumpf, R. Boreth Eam,SarahGregory Parker, Chen, Fish, VikasJoan S. Goel, K. na Adi firsttargetsa eFT226, inhibitor eIF4A, of class in FGFR1/2 drivencancersHER2 and amplifications P HER of a 1 assessed 1 RAS/MAPK of FGFR RTK transiently 1 , / engagement malignancies 2 sequence : 2 FGFR mRNA 5 eIF4B 2 eFT nude eFT and consisting : mTOR amplifications along and ’ patient m P TSC PI3K S6K1 resulted - dependent and of AKT 1 . RTKs 5 UTR and 7 structure in of the G on / ’ and : : 226 226 P eIF4G - 2 2 and eIF4A apoptosis Mutations UTR eFT FGFR translation polypurine or or [eIF MAPK in or these potential . the HER regression confirming of inhibits The 226 transfected is target HER survival NOD/SCID subsets specific and vitro HER 4 dependency P RSK 1 PDCD4 . demonstrates in models mRNA of 5 A1 2 regulated , have ’ RTKs in , FGFR 2 - antitumor is oncogenic 10 ternary eIF expression UTR and - 2 Therapeutics, San Diego, San CA Therapeutics, eFT driven RAS enabling suggesting efficacious the PI by - or treated mRNA , the alterations to 45 . 4 MEK ERK RAF initiation initiated 3 Degradation during Oncoprotein A, translation 2 translational . - 226 leading K/AKT amplifications recognition AKT - use proliferation at 5 the or fold Treatment mice translation eIF ’ with - complex tumors untranslated by HER mRNA] well these was 4 many response signaling, drivers with 5 greater E, clinical was the eFT ’ . were . - the luciferase to signaling 2 that against UTR . and eFT , evaluated tolerated . 226 downregulation potential dose and eFT eFT analyzed Furthermore, . The of with eukaryotic . expression 226 Formation eIF implanted eFT repressor of with elements dependency Solid • • • sensitivity development and 226 226 affecting treated which observed to FGFR 4 tumor polypurine dependent potent 226 is G eIF region tumor component eIF that RTK multiple initiation sequence eFT eFFECTOR mediated reporter pathways . target eFT a resulted apoptosis tumor selectively eIF 4 doses for using by 1 first 4 could A 226 , . are 226 A 1 4 models with enhance western FGFR of is eFT receptor with eFT translation A inhibition ( in and to signaling 5 genes in this . tightly in functions RNA cell this ’ cell , xenograft of - known . 226 in element be In constructs inhibition 226 inhibition UTR) also eFT preclinical in class the 2 Clinical subcutaneous polypurine a key RTK - thereby assays translation data selective breast, and based ternary solid Therapeutics effective with significant proliferation, complex 226 in as inhibit blot increases controlled potent, tyrosine decreased of 5 inhibitor expression the pharmacodynamic eIF ’ of HER oncogenic oncogenes - provides administered of to alterations initiation UTR mRNA . is tumor trials models analysis luciferase of by treatment cell For activates catalyze 4 complex highly non containing in 2 promoting dysregulated A in RTK elements through eFT kinases vivo in inhibitor leading in the - activity the in treating proliferation that at small facilitating xenograft cell small 226 . RTK vivo protein that harboring is by patients a enriched vivo Antitumor the factor drivers, models the affinity in means blocks reporter converts has eFT lines an of survival compared (RTKs) Q formation tumor protein level cell FGFR involved the within translation unwinding experiments, 4 to 226 solid tumor cancer pathways D expression regulates molecule designed essential markers 4 of models with driven oncogene 5 IV driven ribosome lung F in including to 1 of between selective FGFR ’ resulted activate - . , and activity growth tumors UTR eIF assays. (eIF eIF the the FGFR mRNA select levels types solid and to 4 of and 1 cell 4 4 A1 5 5 an by by / of of of of in F) ’ A ’ 2 2 a a - - . GGCGGC, treated with translation B of complexes surface Figure analysis for Figure binding FGFR HEK B. A. D. C. A. ) Results Schematic eIF 24 293t Inhibition eFT226of TargetGenes is Mediated Through the 5’ 2 6 4 constructs - 2 hr A ( 1 mer . ∆ . sites 1 in with . 0 UTR) cells Red and Protein to O eFT formed the by sequence eFT226 SelectiveeFT226 TranslationalSequence ais Regulator specific of : were eFT226 ( 25 N increasing FGFR 226 in CCGCCG wt eFT and presence the O Binding to to Binding containing SNU expression 226 is 2 deleted 100 on treated ternary HO nM , SNU - HER a 16 HO O motif ) is different and sequence concentrations cells AGAGAG sequence 2 - (green) 0 . 16 FGFR2 ( FGFR2 , Black from complex with 5 Cyclin A repeats of ’ and - eFT226 ( N representative UTRs 25 RTKs RNA surface : RNA 5 ∆ increasing CAACAA CN or C ’ selective UTR) D - dependent ) UTR 1 interactions of 100 nM HER are were absence , sequences Tubulin ) FGFR ( of regulated 2 ∆ . FGFR2 Cyclin D1 Cyclin Tubulin UTR) ( IC transiently ∆ eFT translational concentrations 1 50 UTR) Biacore / . (black) or 2 values 226 Luciferase , constructs B. C. [eIF is Vinculin HER in by AGAGAG CCGCCG GGCGGC CAACAA Sequence Sequence summarized AGAGAG GGCGGC CAACAA CCGCCG or 4 5’ MDA A sensogram are 2 eFT RNA RNA - of transfected UTR UTR - hippuristanol eFT or 226 summarized eFT regulator - 0 protein . reporter MB 226- TUBA of Overexpression 226 eIF4A1 through - eFT226 ( 13.8 IC 361 218 1.5 30 eFT eFT226 92 mRNA] 0.021 wt 50 w/ eFT226 3.19 2.43 ( of Sequence right were 9.6 K ( ± ± ( MDA levels 226 ± ± nM 5’ FGFR2 FGFR1 left . 18 0.4 D into TUBA cells HER2 55 2.0 gene eIF - ( 100 nM UTR UTR eFT226 ± ± ± ± µ ) A for 0.001 0.4 0.03 0.01 in M) M) the . 4 ) for panel ) panel . transiently C - the A AGAGAG the Cells were Chemical MB ) Rel. to to Rel. 1 constructs 4 5 61.5 eFT 145 9.2 4 0 ’ binding 1 of - table MDA hr HER2 (∆ HER2 - ) ) UTR hr . 361 . w/o eFT226 were IC 226 . 3.27 3.78 analyzed eFT226 5’ RTK D . The K 8.0 8.0 50 eFT226 ( Green 3’ 4.2 1.7 0.8 30 D ) A/G 36 B . - ( ( ( A/G MB nM IC A - enhances right ± ± ± ± µ Inhibition structure C) transfected UTR) ∆ treated 0.9 0.06 0.3 0.05 ) to mRNA M) M) Hipp 50 stability 210 369 204 426 containing UTR Luciferase ) - ( 100 : 231 Predicted nM nM an panel . by AGAGAG, ) ) AGAGAG constructs cell western Change with - HER2 Vinculin Cyclin D1 Cyclin AGAGAG the Fold Fold 0.34 UTR of 381 2.5 1.6 of ) of Rel. to to Rel. . 0.5 0.9 0.5 line 1 into reporter eFT 5 in binding ternary eFT eFT ’ - Blue UTRs vitro . 226 RNA blot and 226 226 : the B : ) eFT226 DownregulatesRTK Protein ExpressionAKT/MAPK and Signaling protein qPCR panel survival. in Figure Figure blot lines proliferation levels dependent C. A. H. F. D. B. A. NCI analysis with ) analysis - . H results 4 3 eFT levels . 716 . eFT A eFT eFT 1 2 3 4 5 6 7 8 9 10 9 8 7 6 5 4 3 2 1 226 cell ) . tumor 226 Polysome (measured D 226 in 226 of MDA in 40S - lines Vehicle MDA inhibits E BT474 MFM223 MDAMB231 MDAMB361 NCIH1581 NCIH716 NCI 60S mRNA eFT226 TreatmentInhibitsRTKExpression ) RTK Vehicle inhibition Cell line 0 for RTK

translationally 80S tissue NCI Fraction - - NCI MB treated H - MB 10 24 MDA MDA protein 1581 - and profile Polysomes - H1581 (FGFR1 H1581 the from - 0.3 mg/kg H716 (FGFR2 H716 by 361 hr - 30 24 361 (HER2 - - MAPK decreases MB MB tumor translation Cell of with hr 100 Breast Breast Breast Breast Non Colorectal polysome eFT226 (1mg/kg) levels of eFT226 - - after cell Tumor type 361 361 Titer - eFT226 ( eFT small cell lung small cell HER2 MDA p ERK p GAPDH AKT regulates or tissue - - 1 mg/kg 1 AKT S473 ERK amp growth in a 226. AKT - amp Glo) amp RTK Gerson single - vivo ) MB of fractions nM ) 24 ) signaling ) HER protein - and . 361 hr A key and 1 FGFR1 GAPDH - p FGFR2 Tubulin Akt 2 post mg/kg B - apoptosis Akt ) FGFR2 FGFR1 FGFR1 FGFR2 cells . ( HER2 HER2 oncogenic RTK center Western an B levels, S473 - are HER2 dose C - ) treated induction G. E. C. dose Gurwitz Treatment co Proliferation panel after - AKT (measured regulated GI blot D. B. of drivers 50 10.9 11.9 4.4 3.1 5.7 8.4 with eFT ) and ( 20 nM) analysis but of 226. days of MAPK 20 apoptosis NCI not resulting . MDA F , Maria Barrera, Eric Sung, Jocelyn Staunton, Gary G. Chiang, Chiang, G. Staunton,Gary Jocelyn Barrera,Maria Sung, Eric , 0 by nM C - of G - Apoptosis - of H716 (FGFR2 H716 GAPDH D IC ) Q Caspase 10 signaling - ) Down MB 50 eFT FGFR 4 Western 15 16 90 23 NCI NCI 2 6 D ( nM) 25 . - in 226 eFT 361 In vivoIn H - - 2 ( H716 H716 inhibition ) regulation right 50 - and 226 Glo Tabulated or ( as B Apoptosis blot 100 GAPDH ) amp control assessed p treatment. or panel 3 Emax - / 1.6 AKT ) 10 14 eFT226 ( 8 3 4 7 analysis p FGFR2 ERK AKT Tubulin p NCI ) - - ERK AKT S473 of values of ) protein - H as for summary growth RTK nM 716 by shown 3 of ) western for protein hr ( FGFR C levels ) ( and RTK cell left by of 1 • • • • • growth driven seen Treatment 28 inhibition Figure results xenograft xenograft eFT226 TreatmentResultsTumor in Growth Inhibition Regression and Conclusions D. B. A. days Clinical inhibition have eFT eFT motifs stable eFT expression eFT translation by xenograft 5 in . inhibition 226 226 226 226 . eFT the - - tumor after eFT bearing bearing initiated of 226 body ternary 226 NCI inhibits trials is is 20 MDA translationally treatment growth NCI . models - well and H animals inhibits days animals weight a 716 (e. - - H716 (CRC: FGFR2 (CRC: H716 MB with potent Tumor Volume Tumor Volume . - [NCT g regression D) (FGFR inhibition tolerated for - complex . tumor 361 (BC: HER2 361 (BC: were , with the measurements Tumor results the FGFR 2 226 eFT shareholders PAT, 04092673 amp growth treated the indicated growth ) and NPY, in and cell xenograft indicated 1 significant CRS, / [eIF and amp regression down of 2 in Q at amp proliferation selective in 4 inhibition BE, eFFECTOR ) RTK doses taken D and ) RTK 4 VKG, ] with - patients shows bearing A doses tumor driven - JC, of throughout eFT Therapeutics driven either HER regulates after SF, values eFT of C. GSP, regression animals 226 xenografts 226 significant eFT inhibitor 2 15 vehicle AG ) with at . 226 days - xenograft - (Q and through G, mRNA] day the 4 MB, with D Q . or ( C 4 14- solid left . IV) tumor ES, oncogenic induces ) D in eFT NCI Treatment the MDA 15 JS, results panel vivo Dotted of 226 GGC, - in in H1581 (NSCLC: FGFR1 H1581 indicated with growth tumor the - . RTK models MB eIF IV vivo ) CJW, Tumor Volume . Body Weight in A ( line eFT - ) 1 apoptosis (FGFR 361 (BC: HER2 361 (BC: 4 significant of SS, MDA formation mg/kg specific B133 A 226 study tumor KRW, NCI indicates doses 1 malignancies RTK 1 - - is MB , H SHR dependent FGFR or well ( 1581 right of - 361 are 0 tumor . eFT 100% protein 1 growth 2 tolerated 5 employees amp (FGFR mg/kg) and amp panel ’ (HER 226 - ) of UTR ) growth tumor HER 2 1 ) Q . amp amp a and/or for 4 as B 2 D ) ) ) )